melittin-derived peptides for sirna delivery: mechanisms

Melittin-Derived Peptides for siRNA Delivery: Mechanisms of Efficient Cytoplasmic ReleaseArts & Sciences Electronic Theses and Dissertations Arts & Sciences
Spring 5-15-2015
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Recommended Citation Hou, Kirk Kohwa, "Melittin-Derived Peptides for siRNA Delivery: Mechanisms of Efficient Cytoplasmic Release" (2015). Arts & Sciences Electronic Theses and Dissertations. 450. https://openscholarship.wustl.edu/art_sci_etds/450
Division of Biology & Biomedical Sciences Computational and Molecular Biophysics
Dissertation Examination Committee: Samuel Wickline, Chair
Elliot Elson Kathleen Hall
Phyllis Hanson Katherine Henzler-Wildman
by
Kirk Kohwa Hou
A dissertation presented to the Graduate School of Arts and Sciences
of Washington University in partial fulfillment of the
requirements for the degree of Doctor of Philosophy
May 2015
Acknowledgements ....................................................................................................................... vii
Abstract ........................................................................................................................................ viii
Chapter 1: Introduction ............................................................................................................... 1 1.1 Nanomedicine ....................................................................................................................... 1 1.2 RNA Interference by siRNA ................................................................................................ 2
1.2.1 Barriers to the Therapeutic Use of siRNA .............................................................. 4 1.3 Nanoparticles for siRNA Delivery ....................................................................................... 6
1.3.1 Challenges Facing Nanoparticle-Mediated siRNA Delivery: Design Criteria ...... 7 1.4 Current siRNA Delivery Technology ................................................................................... 8
1.4.1 Viral Vectors ........................................................................................................... 8 1.4.2 Cationic Lipids ........................................................................................................ 9 1.4.3 Cationic Polymers ................................................................................................... 9
1.5 Cationic Peptides for siRNA Transfection ......................................................................... 10 1.5.1 Covalent Formulations ......................................................................................... 11 1.5.2 Non-Covalent Formulations ................................................................................. 11 1.5.3 Non-Covalent Formulations: MPG ...................................................................... 13 1.5.4 Non-Covalent Formulations: CADY ..................................................................... 13 1.5.5 Non-Covalent Formulations: dsRBD .................................................................... 14
1.6 Melittin as a Basis for Endosomal Escape ......................................................................... 14 1.7 References .......................................................................................................................... 17
Chapter 2: Novel Melittin-Derived Peptides for siRNA Transfection ................................... 26 2.1 Abstract .............................................................................................................................. 26 2.2 Introduction ........................................................................................................................ 27
2.2.1 Modified Peptides for siRNA Transfection ........................................................... 28 2.3 Materials and Methods ....................................................................................................... 31
2.3.1 Preparation of Peptide/siRNA Nanoassemblies and Analysis .............................. 31 2.3.2 Analysis of p5RHH Disulfide Bond Formation .................................................... 31 2.3.3 Cell Culture ........................................................................................................... 32 2.3.4 siRNA Transfection ............................................................................................... 32 2.3.5 Plasmid DNA Transfection ................................................................................... 33 2.3.6 Western Blotting .................................................................................................... 33 2.3.7 Real-Time PCR ..................................................................................................... 33 2.3.8 Flow Cytometry ..................................................................................................... 34 2.3.9 Cell Viability Assay ............................................................................................... 34
iii
2.4 Results ................................................................................................................................ 34 2.4.1 Screening for siRNA Knockdown .......................................................................... 34 2.4.2 Optimization of Nanoparticle Formulation .......................................................... 35 2.4.3 Nanoparticle Characterization ............................................................................. 37 2.4.4 p5RHH Dimerization Decreases Transfection Efficiency .................................... 41 2.4.5 Comparison with Lipofectamine 2000 .................................................................. 41 2.4.6 p5RHH Performance in the Presence of Serum ................................................... 42 2.4.7 Transfection of Plasmid DNA ............................................................................... 45
2.5 Discussion .......................................................................................................................... 45 2.6 References .......................................................................................................................... 49
Chapter 3: p5RHH Enables pH-Triggered siRNA Release and Endosomal Disruption ..... 54 3.1 Abstract .............................................................................................................................. 54 3.2 Introduction ........................................................................................................................ 55 3.3 Materials and Methods ....................................................................................................... 57
3.3.1 Preparation of Peptide/siRNA Complexes ............................................................ 57 3.3.2 Cell Culture ........................................................................................................... 57 3.3.3 Uptake Inhibition by Flow Cytometry ................................................................... 57 3.3.4 Confocal Microscopy ............................................................................................ 57 3.3.5 Analysis of GFP Knockdown ................................................................................ 58 3.3.6 siRNA Dye Accessibility at Low pH ...................................................................... 58 3.3.7 pH-Dependent Gel-Mobility Assays ..................................................................... 58 3.3.8 Acridine Orange Staining for Lysosomal Disruption ........................................... 58 3.3.9 RBC Hemolysis ..................................................................................................... 59
3.4 Results ................................................................................................................................ 59 3.4.1 p5RHH/siRNA Nanoparticle Endocytosis via Macropinocytosis ......................... 59 3.4.2 Importance of Endosomal Acidification in p5RHH-Mediated Transfection ........ 60 3.4.3 Acidic pH Triggers Nanoparticle Disassembly .................................................... 63 3.4.4 Release of Free p5RHH at Low pH Lyses Membrane-Bound Vesicles ................ 65 3.4.5 Nanoparticle Disassembly Enables siRNA Delivery to the Cytoplasm ................ 65
Chapter 4: Therapeutic Potential for Melittin-Derived siRNA Nanocarriers ...................... 76 4.1 Abstract .............................................................................................................................. 76 4.2 Introduction ........................................................................................................................ 78 4.3 Materials and Methods ....................................................................................................... 79
4.3.1 Cell Culture ........................................................................................................... 79 4.3.2 siRNA Transfection ............................................................................................... 79 4.3.3 Western Blotting .................................................................................................... 80 4.3.4 Real-Time PCR ..................................................................................................... 81 4.3.5 Cell Viability Assay ............................................................................................... 81
iv
4.3.6 Tube Formation Assay .......................................................................................... 81 4.3.7 HUVEC Migration Assay ...................................................................................... 81 4.3.8 Foam Cell Formation Assay/Oil-Red O Staining ................................................. 82 4.3.9 Animal Experiments .............................................................................................. 82
4.4 Results ................................................................................................................................ 83 4.4.1 STAT3 siRNA Delivery to Slow Melanoma Growth ............................................. 83 4.4.2 STAT3 siRNA Delivery to Decrease Angiogenesis ............................................... 83 4.4.3 JNK2 siRNA Delivery to Decrease Foam Cell Formation ................................... 85 4.4.4 siRNA Delivery to Block NFκB Signaling in ATLL .............................................. 89
Chapter 5: Conclusions and Future Directions ........................................................................ 97 5.1 References ........................................................................................................................ 102
Appendix I: Stimulation of Transvascular Transport for Improvement of Nanoparticle Biodistribution ........................................................................................................................ 103 AI.1 Abstract .......................................................................................................................... 103 AI.2 Introduction .................................................................................................................... 104 AI.3 Materials and Methods ................................................................................................... 106
AI.3.1 Synthesis of myr-SIRK ....................................................................................... 107 AI.3.2 HUVEC Culture and Monolayer Preparation .................................................. 107 AI.3.3 Monolayer Resistance Measurements ............................................................... 108 AI.3.4 Caveolin-1 Western Blotting ............................................................................. 108 AI.3.5 Transcytosis Stimulation ................................................................................... 108 AI.3.6 Direct Transcytosis ........................................................................................... 109
AI.4 Results ............................................................................................................................ 109 AI.4.1 Stimulation of Caveolin-1 Phosphorylation ...................................................... 109 AI.4.2 No Adverse Effects on HUVEC Monolayer Integrity ........................................ 110 AI.4.3 Increased Transcytosis of Caveolae-Targeted and Non-Targeted Cargo ........ 111 AI.4.4 Stimulation of Antibody Transcytosis ................................................................ 113
AI.5 Discussion ...................................................................................................................... 113 AI.6 References ...................................................................................................................... 117

List of Figures
Figure 1.1 RNAi Pathways ............................................................................................................. 3 Figure 2.1 Screening of Melittin Derivatives ............................................................................... 36 Figure 2.2 Optimization of Peptide to siRNA Ratio ..................................................................... 38 Figure 2.3 Temporal Evolution of p5RHH/siRNA Nanoparticles ................................................ 40 Figure 2.4 Deep-Etch Electron Microscopy of p5RHH/siRNA Nanoparticles ............................ 40 Figure 2.5 Peptide Dimerization Alters Transfection Efficiency ................................................. 41 Figure 2.6 FACS Comparison of p5RHH and Lipofectamine 2000 ............................................. 43 Figure 2.7 Comparison of p5RHH and Lipofectamine 2000 GFP Knockdown and Cytotoxicity 43 Figure 2.8 Albumin Stabilizes p5RHH/siRNA Nanoparticles ..................................................... 44 Figure 2.9 p5RHH Mediates Plasmid DNA Transfection into HEK293 Cells ............................. 46 Figure 3.1 p5RHH/siRNA Nanoparticle Uptake is Temperature Sensitive ................................. 61 Figure 3.2 Uptake Inhibition Studies ............................................................................................ 61 Figure 3.3 p5RHH/siRNA Nanoparticles Co-localize with FITC-Dextran .................................. 62 Figure 3.4 Endosomal Acidification is Critical for p5RHH-Mediated Transfection .................... 63 Figure 3.5 pH Mediates p5RHH/siRNA Nanoparticle Disassembly and Endosomolysis ............ 66 Figure 3.6 p5RHH Mediates Both siRNA Release and Endosomal Escape ................................. 67 Figure 4.1 Knockdown of STAT3 in B16-Cells ........................................................................... 84 Figure 4.2 Knockdown of STAT3 in HUVECs ............................................................................ 86 Figure 4.3 STAT3 Knockdown in HUVECS Decreases Angiogenesis in vitro ........................... 87 Figure 4.4 JNK2 Knockdown in Macrophages Decreases Foam Cell Formation ........................ 88 Figure 4.5 NFκB Blockade Decreases ATLL Viability ............................................................... 90 Figure 4.6 p5RHH Delivers Cy5.5-Labeled siRNA to Tumors and Kidneys .............................. 90 Figure 5.1 Mechanism of Cytoplasmic siRNA Delivery by p5RHH ........................................... 98 Figure AI.1 HUVEC Monolayer Culture .................................................................................... 107 Figure AI.2 myr-SIRK Stimulates Caveolin-1 Phosphorylation ................................................ 110 Figure AI.3 myr-SIRK Stimulation Increases Caveolar Transcytosis ........................................ 111 Figure AI.4 myr-SIRK Stimulates Antibody Transcytosis ......................................................... 112
vi
List of Tables
Table 1.1 Cell-Penetrating Peptides for siRNA Transfection ....................................................... 12 Table 2.1 Modified Peptide Vectors ............................................................................................. 30 Table 2.2 Sequences Screened for siRNA Transfection ............................................................... 36 Table 2.3 Nanoparticle Characterization ...................................................................................... 38 Table 2.4 Characterization of Albumin-Coated Nanoparticles ..................................................... 44
vii
Acknowledgements
This work would not have been possible without the guidance and support of many
people. I would first like to acknowledge my advisor Dr. Samuel Wickline, whose curiosity and
analytical mindset have been the foundation for an exciting environment in which to do my
thesis work. Additionally, I have great appreciation for the input of our collaborator Dr. Greg
Lanza for his insight and encouragement. During my time in lab, I have had the opportunity to
work in a collaborative environment alongside many talented scientists who have made my
experience more enjoyable. I would particularly like to thank past lab members Dr. Megan
Kaneda and Dr. Kristin Bibee for their guidance and experimental input. Their assistance not
only aided the progress of my research but also helped me to grow as a scientist. I would also
like to thank my committee members who have been incredibly supportive, and more
importantly, have pushed me to think deeper about science in general. I would also like to
acknowledge financial support from the Sigma-Aldrich Pre-Doctoral Fellowship. Finally, I
would like to thank my family, who have offered unwavering support.
viii
by
Doctor of Philosophy in Biology & Biomedical Sciences (Computational and Molecular Biophysics)
Washington University in St. Louis, 2015
Professor Samuel Wickline, Chairperson
The development of small molecules as therapeutic agents for targeted disease treatment
is unable to keep up with the rapid expansion of databases cataloguing disease-causing proteins
enabled by high throughput analysis of patient samples. As an alternative, the use of small
interfering RNA (siRNA) provides a simple means of efficiently and specifically silencing the
expression of these pathogenic proteins without the need for screening and development required
of small molecules. Unfortunately, the delivery of siRNA is not trivial, and existing technology
is characterized by either poor siRNA transfection efficiency or induction of cytotoxicity.
Detailed work by many groups has supported the finding that low transfection efficiency is often
attributable to endosomal entrapment. This phenomenon prevents siRNA from accessing the
cytoplasmic compartment where it is active. Consequently, development of new siRNA vectors
is required to safely promote endosomal escape and delivery of siRNA to the cytoplasm. This
work focuses on the development and characterization of peptides derived from melittin, the
membrane-lytic component of honeybee venom, for siRNA delivery. The most active melittin
derivative, p5RHH, includes modifications to decrease cytotoxicity, increase siRNA binding, and
ix
allow triggered siRNA release in response to the acidic environment encountered during
endocytosis. These peptides bind siRNA to form nanoparticles of 50 to 200 nm in diameter with
a positive zeta potential (+12 mV). This low magnitude surface charge cannot stabilize the
particles against flocculation, necessitating a subsequent coating with serum albumin to achieve
a stable formulation. p5RHH-mediated transfection is characterized by an IC50 in the range of
25 to 100 nM without cytotoxicity at all tested doses. Furthermore, the activity of p5RHH is
attributed to efficient endocytosis via macropinocytosis with nanoparticle disassembly in the
endosome. Particle disassembly releases both siRNA and peptide, allowing p5RHH to disrupt the
endosomal membrane and siRNA to access the cytoplasmic compartment. To demonstrate broad
transfection potential, p5RHH-mediated transfection has been utilized to treat in vitro models of
cancer, angiogenesis, and atherosclerosis. Furthermore, in vivo studies demonstrate the ability of
p5RHH/siRNA nanoparticles to deposit in tumors with little accumulation in clearance organs
such as the liver and spleen. Instead, these studies reveal siRNA clearance via the kidney. Our
results indicate that melittin can be modified for efficient and safe nanoparticle-mediated siRNA
transfection, potentially enabling the clinical use of siRNA. Moreover, our analysis of p5RHH’s
mechanism of action provides a framework to guide the future development of peptide vectors
for siRNA transfection.
1.1 Nanomedicine
The National Institutes of Health define nanomedicine as “an offshoot of
nanotechnology, [referring] to highly specific medical interventions at the molecular scale for
curing disease or repairing damaged tissues,…”1 This definition highlights the potential of
improved medical treatments by leveraging the unique properties of nanoscale materials. For
example, nanoparticles with diameters less than 1,000 nanometers are expected to provide
improvements over existing medical technology by enhancing the performance of known drugs
and enabling the use of previously bio-incompatible therapeutics.2-6 These highly desirable
characteristics have made nanoparticle development a focus of research since polyacrylamide
nanoparticles were first published in the 1970s.7
Early work in the field of nanoparticle-mediated drug delivery has established that the
ability of nanoparticles to improve drug performance stem from their inherent ability to alter a
drug’s interaction with the body. Notably, nanoparticles improve the pharmacokinetics and
biodistribution of the drugs they carry.8, 9 On the most basic level, nanoparticles enable the use of
potentially beneficial drugs that have been discarded due to a lack of biocompatibility. For
instance, insoluble drugs that are unable to be delivered via oral or parenteral means can be
packaged inside of a carrier nanoparticle for delivery into the body.10
Perhaps of even greater utility is the ability of nanoparticles to enable “targeted delivery,”
a drug delivery paradigm which is characterized by delivery of a therapeutic to a specific
location within the body.11 Examples include delivery of antibiotics to infected tissues,
chemotherapy to tumors, or even anti-inflammatory agents to sites of inflammation.2, 5, 12, 13 By
taking advantage of this property, drugs that have a poor therapeutic index due to systemic side-
2
effects become clinically relevant because they no longer enter non-target tissues. For instance,
liposomal formulations of the chemotherapeutic, doxorubicin, not only prevent the cardio-
toxicity associated with free doxorubicin by decreasing deposition in cardiac tissue but also
improve the ability of doxorubicin to exert anti-tumoral effects.14 This example highlights the
ability of nanoparticles to improve drug delivery to diseased tissue while minimizing the
systemic dose for increased therapeutic benefit. Based on these principles, over 40 nanoparticle
formulations have been approved for clinical use to date, indicating that nanoparticle-based drug
delivery has already begun to impact the practice of medicine.15
1.2 RNA Interference by siRNA
RNA interference (RNAi) refers to an evolutionarily conserved mechanism for post-
transcriptional control of protein expression in which short double-stranded RNA (dsRNA)
target specific messenger RNA (mRNA) for degradation, thus preventing protein translation.16
This evolutionarily conserved mechanism is thought to protect against viral infection in yeast
and regulate cellular processes in higher eukaryotes.17 In the context of cellular regulation, the
RNAi machinery is designed to utilize short, 21 to 25 nucleotide, double-stranded micro RNA
(miRNA). miRNA are originally encoded in a long primary RNA which is processed in the
nucleus by Drosha to produce a hairpin-structured primary miRNA (pri-miRNA). pri-miRNA are
then exported from the nucleus for final processing by the RNase III endonuclease DICER
before loading onto the RNA-induced silencing complex (RISC).18 Loaded miRNA subsequently
target the RISC to mRNA through complementary base pairing (Figure 1.1).19, 20 Notably,
mRNA targeting via miRNA only requires partial base pairing by the miRNA sequence. The
3
resulting RNAi is traditionally thought to be a result of translational repression, but in some
cases, miRNA can induce mRNA cleavage by the RISC’s argonaute-2 subunit.20, 21
Although RNAi in eukaryotes is natively initiated by the production of endogenous
miRNA, Tuschl et al. have demonstrated that RNAi can be artificially induced by the delivery of
exogenous small interfering RNA (siRNA) into the cytoplasm of mammalian cells.22 siRNA are
short 21 to 23 base pair duplex RNA oligonucleotides with 5’-phosphorylated ends and two-
nucleotide 3’ overhangs similar to the structure of endogenous miRNA. The “antisense” strand
shares sequence complementarity to a target mRNA, while the “sense” strand serves as a
bystander. When delivered into the cytoplasm of a cell, siRNA can co-opt the native RNAi
machinery and induce assembly of the RISC. The RISC unwinds siRNA, binds the antisense
strand, and cleaves the sense strand.23 This allows the antisense strand to base pair with target
Figure 1.1 RNAi Pathways Endogenous RNAi is induced by miRNA, which results in translational repression. Alternatively, exogenous siRNA can induce RNAi by mediating mRNA degradation.
4
mRNA. In contrast to miRNA-based mRNA silencing, siRNA are designed to base pair
completely with the desired mRNA and function solely by inducing mRNA cleavage (Figure
1.1).24 This catalytic mechanism allows one siRNA to induce degradation of multiple mRNAs
and leads to efficient, prolonged downregulation of protein expression for 5 to 7 days. Indeed,
RNAi induced by siRNA is only limited by its dilution through cell division.25
The ability of exogenously delivered siRNA to silence protein expression has limitless
potential for the realization of personalized medicine. By silencing the expression of any
combination of proteins involved in disease pathogenesis on an individual basis, a patient’s
particular disease processes can be specifically inhibited with few off-target toxicities. Notably,
siRNAs can be developed against these targets without the lengthy lead identification and
optimization required of small molecule drug development. Given the advantages of siRNA as a
potential therapeutic, there has been a dramatic push to translate siRNA from a research tool to
clinical applications.26 Recently, clinical trials have been initiated for the use of naked siRNA in
ocular, renal, and hepatic diseases.27
1.2.1 Barriers to the Therapeutic Use of siRNA
There are several significant challenges that need to be resolved before siRNA can be
widely used as a therapeutic. On a cellular level, the mechanism of siRNA action dictates that
siRNA must be delivered to the cytoplasm. This requirement represents the primary hurdle
limiting siRNA’s utility as both a basic science research tool and as a clinical therapeutic.
Unfortunately, siRNA are large (~21 kDa) and highly charged, which hinders its direct
translocation across the hydrophobic core of the cellular membrane.28 Moreover, the barrier
provided by impermeable membrane bilayers not only applies to direct translocation from the
extracellular milieu into the cytoplasm but also cytoplasmic access of siRNA enclosed in
5
endocytic vesicles.29 Entrapment of siRNA in endocytic compartments prevents siRNA from
reaching the cytoplasm and accelerates siRNA degradation by the harsh acidic environment
encountered during endosomal/lysosomal trafficking.30
siRNA delivery in an in vivo setting is further complicated by rapid clearance. First, an
abundance of serum RNases causes siRNA cleavage with a half-life of less than 20 minutes.31, 32
Moreover, the size of siRNA (~7.5 nm) allows naked siRNA to be cleared quickly through the
kidney into the urine, as the glomerular basement membrane has a pore size of 10 nm. Detailed
studies have shown that systemically injected siRNA accumulates in the kidney and is excreted
into the urine within one hour.33 The lability of siRNA in serum and rapid clearance of siRNA by
the kidney lead to a short circulation half-life, preventing siRNA accumulation in diseased
tissue.34
Use of siRNA is further limited by the innate immune system. Specifically, pattern
recognition receptors found in both immune and non-immune cells allow highly sensitive
detection of dsRNA, a telltale sign of viral infection.35 These receptors include Toll-like
Receptor (TLR) 3 on the cell surface, TLRs 7 and 8 in the endosomal/lysosomal pathway, and
Protein Kinase R (PKR) and Rig-I in the cytoplasm.36 Spatial separation of these receptors
allows detection of dsRNA in all compartments for a robust antiviral response, leading to
decreased translation of viral proteins and expression of inflammatory cytokines. Fortunately,
detailed studies regarding the activation of these receptors reveal that synthetic dsRNAs less than
23 base pairs with two-nucleotide 3’ overhangs and minimal GA/GU regions are able to
minimize the induction of innate immune responses.35, 37
Despite the myriad barriers preventing the utilization of siRNA as a therapeutic, siRNA-
based therapy is moving towards clinical application as solutions to bypass these barriers are
6
developed. For example, optimization of siRNA chemistry using phosphorothioate backbones
and 2’O-methyl ribose sugars has improved the resistance of siRNA to serum proteins.38-40 While
these solutions resolve some of the problems associated with siRNA delivery, the most
fundamental difficulties remain unsolved. Without a mechanism to promote siRNA entry into the
cytoplasm, the clinical utility of naked siRNA will be limited.41, 42
1.3 Nanoparticles for siRNA Delivery
The application of nanoparticle technology to siRNA delivery solves many of the
challenges associated with in vivo siRNA delivery. Most importantly, nanoparticles can be used
to package siRNA to improve the pharmacokinetic profile of siRNA. By incorporating siRNA
into a carrier nanoparticle, siRNA are inaccessible to serum endonucleases and thus, protected
from degradation. Moreover, nanoparticles with a diameter larger than 10 nm will exhibit
minimal glomerular filtration and decreased kidney clearance.43 Nanoparticles can also impart
additional benefits on siRNA-based therapy by enabling targeted delivery. Given the potential
for erroneous gene silencing in non-diseased tissue, targeted delivery is critical for increased
patient safety when utilizing siRNA therapies designed to target endogenous genes.44-46 When
considering the challenges associated with systemic delivery of naked siRNA, it appears that the
application of nanoparticle technology is perfectly suited to increase siRNA’s circulation half-
life and bioavailability.
On a cellular level, nanoparticles also prove advantageous for increasing siRNA uptake.
By masking the negative charge of the siRNA backbone, nanoparticles can improve association
with the cell membrane via electrostatic association with negatively charged proteoglycans or by
binding cell surface receptors. Both mechanisms can lead to increased endocytosis and cellular
7
uptake. With these properties, nanoparticles can be considered to be siRNA-carrying Trojan
horses which protect siRNA and induce cellular uptake while avoiding TLR-mediated activation
of the innate immune system.47
1.3.1 Challenges Facing Nanoparticle-Mediated siRNA Delivery: Design Criteria
Despite the initial success of some nanoparticle-siRNA formulations, widespread use of
nanoparticle-siRNA technology is still hindered by limitations such as stimulation of innate
immunity, vascular constraint, clearance via the reticulo-endothelial system (RES), and
endosomal entrapment.48 When injected into the blood stream, siRNA-carrying nanoparticles
encounter plasma proteins including components of the complement cascade. Complement
activation can cause degradation of liposomal carriers or stimulate a systemic inflammatory
response.9, 49 Intravenous administration of nanoparticles also contributes to poor nanoparticle
biodistribution. Specifically, endothelial tight junctions limit the ability of nanoparticles to
extravasate, thus preventing therapeutics from reaching their target site and promoting clearance
by macrophages in the liver and spleen.50 Even when nanoparticles are able to extravasate and
enter the targeted cell, effective siRNA delivery can be limited by inappropriate subcellular
localization. Nanoparticle entry via endocytosis often constrains nanoparticles to the endosomal
pathway, ultimately resulting in siRNA degradation in the lysosomes.51
These challenges have prevented nanoparticles from achieving the therapeutic benefits
often espoused by the field of nanomedicine. To overcome these barriers, the ideal siRNA
delivery vector should meet the following criteria: 1) package siRNA to prevent degradation by
serum endonucleases and minimize glomerular filtration, 2) provide sufficient circulation half-
life to allow delivery to the target organ while avoiding RES uptake, 3) avoid opsonization and
stimulation of an immune response, 4) induce endocytosis via cell surface receptor binding or
8
deliver siRNA to the cytoplasm, and 6) exhibit minimal cytotoxicity.
As nanoparticles have matured, difficulties associated with toxicity, immune stimulation,
and RES-mediated clearance have been addressed through improved nanomaterials. Notably,
criteria two through four apply to in vivo usage, and while they are not trivial, have been solved
by a combination of pegylation and active targeting.8, 52 Pegylation of nanoparticles decreases the
interaction of serum proteins with nanoparticle surfaces via steric hindrance provided by
hydrophilic polyethylene glycol (PEG) polymers. By preventing opsonization, nanoparticles are
no longer rapidly cleared by scavenger receptor-carrying macrophages in the RES. For instance,
cyclodextrin nanoparticles rely on pegylation to decrease opsonization and therefore limit RES
clearance allowing long circulation half-lives.53, 54 These nanoparticles can then slowly penetrate
tumors to deliver therapeutic siRNA to tumor cells via active targeting to the transferrin receptor.
Unfortunately, existing solutions have had difficulties fulfilling criteria five and six, which are
specific to the siRNA delivery vector itself. Existing siRNA delivery technology has traditionally
exhibited efficient endosomal escape with high cytotoxicity or poor endosomal escape with low
cytotoxicity.55, 56 Consequently, there is a need for new siRNA delivery technology to enable
endosomal escape with minimal cytotoxicity.
1.4 Current siRNA Delivery Technology
1.4.1 Viral Vectors
Early work has utilized adenoviral vectors to deliver plasmids expressing short hairpin
RNA (shRNA), which are converted into siRNA via the nuclease DICER. These methods can
achieve highly efficient plasmid transfection and production of high amounts of encoded
9
shRNA.57 However, early clinical trials were halted by excessive inflammatory responses and
induction of cancer due to genomic integration of the delivered plasmid.58-60 These difficulties
suggest that new non-viral siRNA delivery strategies are required to fully realize the promising
therapeutic potential of siRNA.
1.4.2 Cationic Lipids
Cationic lipids are the most efficient and best characterized non-viral vectors for the
delivery of siRNA. The original class of lipid vectors was based on the cationic lipids DOTAP or
DC-Chol mixed with a helper lipid, DOPE, to package siRNA into multi-lamellar lipoplexes.61
Importantly, cationic lipids are also able to induce disruption of endosomal membranes
(endosomolysis) by altering lipid bilayers to favor non-bilayer structures.62 Unfortunately, the
efficiency with which cationic lipids disrupt membrane bilayers also leads to considerable
cytotoxicity.63 A new generation of synthetic lipids (lipidoids) delocalizes cationic charge over a
large headgroup and exhibits both drastically reduced cytotoxicity and increased siRNA
transfection efficiency.64, 65 Unfortunately, in vivo results have not been as successful as those
from in vitro studies owing to heterogeneity in lipid formulations. Nonetheless, existing lipoplex
formulations have reached Phase I clinical trials with moderate success for liver disease.66
However, some similar lipoplex-based therapeutics have been shelved due to stimulation of
systemic immune responses at high doses.67 Although lipoplexes are currently the primary
vectors for in vivo applications, these findings highlight the need for further analysis of lipoplex-
mediated toxicity.
1.4.3 Cationic Polymers
Cationic polymers offer a high charge density with which to condense and package
siRNA. Traditional polymer vectors include synthetic polymers such as polyethyleneimine (PEI)
10
and cyclodextrin, and natural polymers such as the polysaccharide chitosan.68 These polymers
rely on positively charged groups to neutralize and package siRNA to allow for efficient
endocytosis, while utilizing protonatable moieties to induce osmotic lysis of endosomes via the
“proton sponge effect.”69 This method of endosomolysis relies on buffering of endosomal
acidification by weak bases, allowing the ensuing accumulation of chloride counter-ions to drive
osmotic rupture of the endosome. Despite robust endosomolysis, polymer vectors are difficult to
optimize. The degree of optimization must be carefully controlled, as high degrees of
polymerization provide improved siRNA compaction and transfection but also increase
cytotoxicity via production of reactive oxygen species and destabilization of cellular
membranes.55, 70 Despite these difficulties, polyplexes, notably cyclodextrin-based copolymers,
have entered Phase I clinical trials for targeted cancer therapy. Unfortunately, this trial has failed
to move forward, possibly due to low endosomal escape or nanoparticle disassembly on
glomerular basement membranes in the kidney.71-73
1.5 Cationic Peptides for siRNA Transfection
With the observation that the Trans-Activator of Transcription (TAT) peptide from HIV
can directly translocate across cell membranes to trans-activate the viral promoter in tissue
culture, cell-penetrating peptides (CPPs) have become a widely utilized tool for delivery of
therapeutics.74 Delivery of therapeutics ranging from small molecules to proteins have all been
augmented by CPP technology.75 Based on their hypothesized ability to bypass the cellular
membrane, CPPs were expected to enable cytoplasmic delivery of siRNA while avoiding the
endosomal compartment. Although this has ultimately proven untrue, peptides remain a viable
11
option for siRNA delivery due to an ability to promote endocytosis and a relative lack of
cytotoxicity.
1.5.1 Covalent Formulations
Initial attempts to harness peptides for siRNA transfection focused on direct chemical
conjugation to known cell-penetrating peptides such as penetratin and transportan. These studies
reported an IC50 of 25 nM and minimal cytotoxicity.76-78 Unfortunately, these initial studies
were later shown to be confounded by poor purification, as excess unconjugated peptide
augmented the transfection efficiency. Turner et al. demonstrated that after careful purification,
siRNA conjugated to cell-penetrating peptides had minimal transfection capacity, requiring a
concentration of 5 µM to achieve significant knockdown.79-81 They attributed the limited
transfection capacity to endosomal entrapment based on the findings that endosomolytic agents
such as chloroquine were able to release siRNA into the cytoplasm.82 When tested for intra-
tracheal delivery of siRNA to the lung, these purified conjugates did not exhibit any
improvement over naked siRNA alone.83 Additional setbacks included decreased peptide-siRNA
conjugate uptake in the presence of serum proteins.84 Nonetheless, in vitro studies investigating
the use of peptide-oligonucleotide conjugates reinforce the safety of peptide vectors. Only
minimal cytotoxicity has been reported with doses up to 10 µM, indicating a high degree of
safety on a cellular level.85
1.5.2 Non-Covalent Formulations
Due to the limited efficacy of peptide-siRNA conjugates and the finding that excess
peptide imbues improved transfection efficiency, peptide-based siRNA vectors have more
commonly been utilized in non-covalent formulations (Table 1.1). Initial studies examining
siRNA delivery via electrostatic packaging by TAT, penetratin, and transportan all produced
12
Table 1.1 Cell-Penetrating Peptides for siRNA Transfection Peptide Target Gene IC50 Ref TAT47-57 (YGRKKRRQRRR) eGFP none 81, 86 Penetratin (RQIKIWFQNRRMKWKK-amide) Luciferase none 87-89 Transportan (GWTLNSAGYLLGKINLKALAALAKKIL-amide) Luciferase none 87 TP10 (AGYLLGKINLKALAALAKKIL-amide) Luciferase none 87, 88 Rn (8<n<15) R9-RVG GFP 100nM 90 R9 VEGF none 88, 91 R15 VEGF 100nM 92 Dermaseptin S4 Luciferase none 93 MPG (Ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-amide) Luciferase >100nM 94
MPGΔNLS (Ac-GALFLGFLGAAGSTMGAWSQPKSKRKV-amide) Luciferase, GAPDH, Cyclin B1 30-50nM 94, 95
MPG8 (Ac-βAFLGWLGAWGTMGWSPKKKRK-amide) Cyclin B1 1nM 95 MPGα (Ac-GALFLAFLAAALSLMGLWSQPKKKRKV-amide) Luciferase 1nM 96 CADY (GLWRALWRLLRSLWRLLWRA-amide) GAPDH <1nM 97-100 dsRBD Luciferase none 101 TAT-dsRBD GFP 400nM 102
minimal siRNA-mediated knockdown despite high levels of siRNA internalization.81, 86, 87
Further treatment with chloroquine increased siRNA-mediated knockdown, revealing that some
of these peptides were unable to achieve sufficient endosomal escape.88 Interestingly, penetratin
and poly-arginine peptides continued to exhibit minimal knockdown in the presence of
chloroquine. This implies that endosomolysis alone is not sufficient for induction of RNAi, but
efficient release from the vector is also required. These initial studies point out the requirement
for peptide vectors to both release siRNA and promote endosomal escape in order to achieve
maximal siRNA transfection efficiency.
Despite these initial difficulties, the development of new peptide sequences has allowed
siRNA delivery with non-covalent formulations. Notably, the transfection efficiency (IC50 less
than 1 nM) of MPG and CADY is much higher than that of covalent strategies (IC50 greater than
5 µM).103-105 Moreover, peptide vectors have demonstrated efficacy in a variety of cell types in
tissue culture as well as successful abatement of cancer progression in mouse models.56
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peptide/siRNA complexes continue to exhibit remarkable safety with minimal toxicity, even at
high µM concentrations. Unfortunately, the limiting factor for non-covalent peptide vectors
appears to be excessive endosomal entrapment as well as decreased transfection in the presence
of serum proteins.98, 106
1.5.3 Non-Covalent Formulations: MPG
Initial success with peptide-mediated siRNA delivery was achieved with MPG, a hybrid
peptide consisting of the fusion sequence from HIV glycoprotein 41 and the nuclear localization
sequence of the SV40 virus. MPG was originally developed for plasmid DNA transfection and
achieves knockdown of target mRNA with an IC50 of 20 to 50 nM.94, 95, 107 Initial work indicated
that MPG-mediated siRNA transfection via direct translocation through the cell membrane.108
However, later studies concluded that MPG/siRNA complexes enter cells via
macropinocytosis.109 Further sequence refinement led to a truncated form, MPG-8, as well as
MPGα, which exhibits increased membrane-inserting properties.95, 96 Both MPG-8 and MPGα
are characterized by sub-nanomolar IC50 when used to target luciferase. However, detailed work
by Veldhoen et al. demonstrated that MPGα is not especially efficient and requires almost two
orders of magnitude more siRNA "per dose" to achieve the same knockdown as Lipofectamine
2000.96 This finding may be attributable in part to endosomal entrapment, as treatment with
chloroquine improved transfection by nearly 20%.
1.5.4 Non-Covalent Formulations: CADY
CADY is the first peptide designed to specifically promote both siRNA binding and
membrane permeability. CADY has an alpha-helical structure with an siRNA-binding face
containing cationic residues and a membrane-binding face enriched in tryptophan residues.99 To
14
date, CADY is the most efficient peptide-based siRNA vector with an IC50 less than 0.5 nM.97
Interestingly, CADY appears to function by direct membrane translocation as neither ATP
depletion nor incubation at 4°C inhibits siRNA transfection.100 Nevertheless, CADY may have
limited in vivo applications due to decreased transfection in the presence of serum proteins.98
1.5.5 Non-Covalent Formulations: dsRBD
TAT and double-stranded RNA binding domains (dsRBD).101, 102 dsRBD sequences were
modified from Protein Kinase R, a cytoplasmic protein which plays a crucial role in the detection
of viral infection. These TAT-dsRBD conjugates were shown to transfect a variety of difficult to
transfect cell lines such as Jurkat T-cells and endothelial cells in tissue culture with an IC50 near
400 nM and were able to yield significant siRNA knockdown in vivo. Unfortunately, later studies
demonstrated that, due to low binding affinities, a single dsRBD is unable to bind siRNA
efficiently, suggesting that siRNA packaging by TAT-dsRBD is attributable to electrostatic
TAT/siRNA interactions.110
1.6 Melittin as a Basis for Endosomal Escape
It is apparent that peptide-mediated transfection is hampered by poor efficiency due in
part to endosomal entrapment. In this work, we employ melittin derivatives for their membrane
inserting properties as a potential solution to enable endosomal release of siRNA. Melittin is a 26
amino acid alpha helical peptide first purified from the European honeybee in 1958 that has a
high affinity for lipid membranes and ultimately causes membrane lysis in its active form.
Although the exact mechanism of membrane disruption has not fully been clarified, melittin has
15
been shown to lyse red blood cells and model membranes.111, 112 Given its lytic nature, melittin
itself has been proposed for the treatment of cancer and bacterial infections.
More recently, melittin has been utilized as an excipient for hepatocyte-targeted siRNA
therapy based on its membrane-lytic properties.113 In these studies, melittin is protected by acid
labile groups that prevent membrane insertion until exposed to the acidic endosomal
environment.114 Activation of melittin in the hepatocyte endosome has proven to be robust and
yields a 500-fold increase in siRNA-mediated protein knockdown in vivo.
Unfortunately, similar studies in which siRNA and melittin are conjugated to a polymer
backbone have not been as successful. These studies have shown that such constructs cause
substantial cytotoxicity in tissue culture and liver necrosis with abdominal bleeding in mice.115-
117 These conflicting results highlight the difficulties of working with melittin as a therapeutic.
Moreover, melittin has been shown to be less lytic at acidic pH, the same environment found in
endosomes.118 Nonetheless, the apparent ability of melittin to promote endosomolysis implies
that melittin may be a potential solution to the problem of endosomal entrapment.114
Melittin contains a hydrophobic N-terminus and cationic C-terminus similar to the
amphipathic sequence of previously published siRNA-transfecting peptides. In studies of
plasmid DNA delivery, melittin was able to bind DNA but yielded poor condensation due to an
inadequate number of basic residues.119 Polymerization of melittin improved DNA condensation
and yielded moderate transfection. These findings suggest that for stable condensation of siRNA,
additional basic residues must be appended to the native melittin sequence. Furthermore, our lab
has previously demonstrated that N-terminal melittin truncations decrease cytotoxicity by two
orders of magnitude while maintaining the peptide’s propensity to partition into lipid
16
membranes.120 By taking advantage of these modifications, melittin derivatives may provide a
starting point for a new class of peptide vectors for siRNA transfection.
Accordingly, we proposed to investigate the following hypotheses:
Chapter 2: Given its amphipathic nature and net positive charge, melittin can be modified to
perform as an siRNA delivery vehicle with minimal cytotoxicity.
Chapter 3: Nanoparticles composed of siRNA and melittin derivatives function by disassembling
in the endosome, releasing free peptide to trigger endosomal escape.
Chapter 4: Melittin derivatives can transfect siRNA into a variety of cell types for the treatment
of clinically relevant disease processes.
17
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104. Hassane, F. S.; Saleh, A. F.; Abes, R.; Gait, M. J.; Lebleu, B., Cell penetrating peptides: overview and applications to the delivery of oligonucleotides. Cell. Mol. Life Sci. 2010, 67 (5), 715-726.
105. Meade, B. R.; Dowdy, S. F., Enhancing the cellular uptake of siRNA duplexes following noncovalent packaging with protein transduction domain peptides. Adv. Drug Del. Rev. 2008, 60 (4– 5), 530-536.
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106. Mäe, M.; Andaloussi, S. E.; Lehto, T.; Langel, Ü., Chemically modified cell-penetrating peptides for the delivery of nucleic acids. Expert Opinion on Drug Delivery 2009, 6 (11), 1195-1205.
107. Deshayes, S.; Gerbal-Chaloin, S.; Morris, M. C.; Aldrian-Herrada, G.; Charnet, P., et al., On the mechanism of non-endosomal peptide-mediated cellular delivery of nucleic acids. Biochimica et Biophysica Acta (BBA) - Biomembranes 2004, 1667 (2), 141-147.
108. Deshayes, S.; Morris, M.; Heitz, F.; Divita, G., Delivery of proteins and nucleic acids using a non- covalent peptide-based strategy. Adv. Drug Del. Rev. 2008, 60 (4–5), 537-547.
109. Veldhoen, S.; Laufer, S. D.; Restle, T., Recent developments in peptide-based nucleic acid delivery. Int J Mol Sci 2008, 9 (7), 1276-1320.
110. Geoghegan, J. C.; Gilmore, B. L.; Davidson, B. L., Gene silencing mediated by siRNA-binding fusion proteins is attenuated by double-stranded RNA-binding domain structure. Molecular Therapy — Nucleic Acids 2012, 1 (11).
111. Bogaart, G. v. d.; Guzmán, J. V.; Mika, J. T.; Poolman, B., On the mechanism of pore formation by melittin. J. Biol. Chem. 2008, 283 (49), 33854-33857.
112. Pratt, J. P.; Ravnic, D. J.; Huss, H. T.; Jiang, X.; Orozco, B. S., et al., Melittin-induced membrane permeability: A nonosmotic mechanism of cell death. In Vitro Cell. Dev. Biol. Anim. 2005, 41 (10), 349-355.
113. Wooddell, C. I.; Rozema, D. B.; Hossbach, M.; John, M.; Hamilton, H. L., et al., Hepatocyte- targeted RNAi therapeutics for the treatment of chronic Hepatitis B Virus infection. Mol. Ther. 2013, 21 (5), 973-985.
114. Rozema, D. B.; Ekena, K.; Lewis, D. L.; Loomis, A. G.; Wolff, J. A., Endosomolysis by masking of a membrane-active agent (EMMA) for cytoplasmic release of macromolecules. Bioconj. Chem. 2003, 14 (1), 51-57.
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117. Meyer, M.; Zintchenko, A.; Ogris, M.; Wagner, E., A dimethylmaleic acid–melittin-polylysine conjugate with reduced toxicity, pH-triggered endosomolytic activity and enhanced gene transfer potential. The Journal of Gene Medicine 2007, 9 (9), 797-805.
118. Tan, Y.-X.; Chen, C.; Wang, Y.-L.; Lin, S.; Wang, Y., et al., Truncated peptides from melittin and its analog with high lytic activity at endosomal pH enhance branched polyethylenimine-mediated gene transfection. The Journal of Gene Medicine 2012, 14 (4), 241-250.
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2.1 Abstract
Traditional transfection agents such as cationic lipids and polymers have high siRNA
transfection efficiency but cause extensive cytotoxicity. Alternatively, CPP-based transfection
agents exhibit improved cytotoxicity profiles but do not have the efficiency of existing lipidic
agents due to endosomal entrapment. As a consequence, we propose an alternative strategy for
efficient peptide-mediated siRNA transfection by starting with melittin, a known membrane-lytic
peptide. Through the incorporation of modifications designed to decrease cytotoxicity and
improve siRNA binding, we have generated a panel of melittin derivatives with the ability to
transfect siRNA. The most active variant, p5RHH, can complex siRNA to form nanoparticles 50
to 200 nm in diameter at peptide to siRNA ratios of 100 to 1. These nanoparticles are stabilized
by weak electrostatic repulsion (zeta potential +12 mV) with loss of activity over time due to
flocculation. Despite particle instability, p5RHH does not induce cytotoxicity and exhibits high
efficiency with greater than 50% GFP knockdown at 50 nM, as determined by RT-PCR.
Moreover, kinetically stabilized formulations can be achieved by coating the particles with
albumin, preventing flocculation for over 72 hours. These data confirm that our strategy for
development of siRNA-transfecting peptides can provide an alternative avenue to safe and
effective siRNA transfection.
NOTE: Portions of this chapter are adapted from previously published work.
Hou, K.K.; Pan, H.; Lanza, G.M.; Wickline, S.A., Melittin derived peptides for nanoparticle
based siRNA transfection. Biomaterials 2013, 34 (12), 3110-3119.
27
2.2 Introduction
RNAi induced by siRNA has been proposed as a highly effective therapy for myriad
diseases including cancer and atherosclerosis.1, 2 However, despite nearly two decades of intense
research since ground breaking work by Tuschl et al. revealed the potential for siRNA in
mammalian cells, siRNA therapeutics have demonstrated limited success in translation to clinical
applications.3, 4 The major barriers preventing successful siRNA-based therapeutics comprise
poor cellular uptake and instability of free siRNA in serum. Its large molecular weight (~21 kDa)
and high charge density prevent siRNA from passing through the cellular membrane to reach the
cytoplasmic compartment where siRNA is active, thus blocking successful induction of RNAi.
These traits, combined with a serum half-life of less than 20 minutes, necessitate the packaging
of siRNA by transfection agents.5 Such agents can protect siRNA from serum endonucleases and
promote siRNA uptake through endocytosis. Unfortunately, endocytic pathways present a second
barrier, as siRNA must escape the endosomal/lysosomal compartment where it is degraded by an
increasingly acidic environment.5-9
Despite these challenges, cationic lipids and polymers have been successfully employed
for siRNA transfection.2, 5, 6, 10-12 Unfortunately, these classes of transfection agents often exhibit
unacceptable cytotoxicity.13-16 The incorporation of cationic lipids into membrane bilayers within
cells promotes siRNA release into the cytoplasm but also causes generation of reactive oxygen
species and Ca+2 leakage, a side effect shared by high molecular weight polyetheyleneimine
cationic polymers.15-17 Despite continued development of these siRNA vectors with the goal of
reducing cytotoxicity, these agents have experienced difficulties when administered in vivo due
to aggregation with serum proteins and complement activation.18-20 If the problem of systemic
siRNA delivery is to be solved, new classes of siRNA transfection agents need to be developed.
28
CPP-based siRNA transfection agents have shown promise with respect to reducing
cytotoxicity.21-25 Although CPP-based siRNA transfection appears nearly free of cytotoxicity,
peptide-based transfection agents have not achieved the high efficiency of traditional lipidic
transfection agents. Some insight has been provided by the studies of Veldhoen et al., which
suggest that peptide-based transfection is limited by lysosomal trapping.26 Despite early work
showing that CPPs mediate siRNA uptake in an energy independent manner,27, 28 it appears that
nanoparticles produced by the assembly of CPP and siRNA enter cells via endocytosis and must
escape the endosomal/lysosomal pathway to gain access to the cytosolic compartment.21, 22, 29, 30
With this barrier in mind, existing CPP technology has achieved a new level of sophistication
through the chemical conjugation of CPPs to membrane-active lipids or endosomolytic agents.24,
31-33 Despite these advances, achieving transfection efficiency comparable to that of lipids has
remained elusive.
Modifications to cell-penetrating peptides for increased endosomal escape have centered
on three modifications: fusion to pH-sensitive fusogenic viral peptides, conjugation to lipidic
moieties, and modification with buffering agents to promote osmotic rupture of endosomes
(Table 2.1).8, 34 Standard CPPs such as penetratin and TAT have been fused with portions of
influenza proteins hemagluttin-2 (HA2), LK15, or N-E5L which mediate viral escape from the
endosome when exposed to acidic pH.35-38 This functional pH-triggered structural change
increases the alpha helical content of the peptide and promotes insertion into the endosomal
membrane. In vitro, HA2 fusions have shown the ability to improve siRNA transfection when
packaged by CPP.35 Unfortunately, this modification increases the cytotoxicity of the peptide
with significant cell death at 1 µM.35 Furthermore, these fusion peptides are not maximally
29
efficient as treatment with chloroquine improves siRNA transfection. This indicates that
endosomal entrapment is a problem that is not completely addressed by these methods.36
Additional attempts to increase the membrane-disruptive potential of cell-penetrating
peptides come in the form of conjugation to lipids, most commonly stearyl moieties.33, 39
Originally developed for plasmid DNA, stearylated peptides also improve the transfection of
small oligonucleotides such as siRNA and splice-correcting oligonucleotides.40, 41 For these
purposes, stearyl-TP10 has proven to be more effective than stearyl-penetratin, stearyl-TAT, or
stearyl-R8. Studies utilizing un-stearylated peptides suggest that differences in these stearylated
variants may be due to the ability of the peptide to release siRNA.42, 43 Moreover, in the case of
stearyl-TP10, authors noted that improved transfection may not be due to improved endosomal
escape but may actually be attributable to improved particle stability and increased siRNA
uptake.42 In fact, treatment with chloroquine doubled siRNA-mediated knockdown, again
revealing poor endosomal release.
The importance of endosomal pH buffering as a release mechanism has led researchers to
take advantage of histidine residues as a potential trigger for endosomal escape.44 Histidine
residues are unique for their ability to be protonated at acidic pH (pKa ~6) while remaining
uncharged at neutral pH. By incorporating high percentages of protonatable histidine residues,
poly-arginine and TAT have been modified to increase their ability to deliver nucleotides to the
cytoplasm. For instance, the addition of ten histidine residues to TAT improves plasmid DNA
transfection by 7000 fold over TAT itself.45
Other researchers have combined these strategies to improve peptide-mediated
endosomal escape. By taking advantage of stearylation and conjugation to the proton-buffering
agent chloroquine, Andaloussi et al. have created Pepfect6, which has an IC50 less than 10 nM
30
in a variety of cell types.46-48 Despite high efficiency transfection of hepatocytes in vivo, further
analysis reveals that Pepfect6/siRNA particles demonstrate an increase in size and a mild
decrease in transfection in the presence of serum proteins.46 Despite these difficulties, the
incorporation of these modifications is indicative of the need for supplementary endosomolytic
agents to improve the endosomal-escape properties of existing CPPs.
In this chapter, we propose an alternative strategy for efficient peptide-based siRNA
transfection based on modification of the cytolytic peptide, melittin, which is the pore forming
component of honeybee venom. Melittin’s ability to form pores in membrane bilayers suggests
that it can serve as a foundation for the development of simple peptides, which can improve
Table 2.1 Modified Peptide Vectors Modified Peptides Benefit Ref TAT Stearylation Improved siRNA uptake/Endosomal escape 40 HA2 conjugation pH-triggered endosomal escape 49 LK15 conjugation Endosomal escape 50 Histidine10 conjugation pH-triggered endosomal escape 45 Penetratin HA2 conjugation pH-triggered endosomal escape 51 Stearylation pH-triggered endosomal escape 42 Poly-Arginine Stearylation Endosomal escape 42, 52 Myristoylation Brain targeted delivery 53 Cholesterol conjugation Increased particle stability/Endosomal escape 54 Stearylation and insertion of histidine Endosomal escape 55 Poly-Lysine Insertion of histidine Endosomal escape 44, 56, 57 TP10 EB1 Endosomal Escape 58 Stearylation Improved siRNA uptake/Endosomal escape 42 Stearylation and chloroquine conjugation pH-triggered endosomal escape 46, 48, 59 MPG8 Cholesterol conjugation Improved particle stability/Endosomal Escape 60 Calcitonin-derived peptides Myristoylation Improved siRNA uptake/Endosomal Escape 36 HA2 conjugation Improved siRNA uptake/pH-triggered endosomal escape 36
31
endosomal escape, thereby setting the stage for efficient siRNA-mediated RNAi. Previous work
in our lab has shown that melittin can be modified to attenuate its cytotoxicity while maintaining
its propensity for interacting with membrane bilayers.61, 62 By incorporating these changes along
with modifications to enhance peptide/siRNA interactions, we hypothesize that melittin-derived
peptides can safely transfect siRNA by improving siRNA delivery to the cytoplasmic
compartment.
Melittin derivatives were synthesized by Genscript (Piscataway, NJ), dissolved at 10 mM
in RNase/DNase free water (Sigma, St. Louis, MO) and stored in 4 µL aliquots at -80C before
use. p5RHH/siRNA transfection complexes were prepared by diluting p5RHH 1 to 200 in
phosphate buffered saline (PBS, Sigma), vortexing for 30 seconds followed by addition of the
appropriate amount of siRNA (stock concentration of 10 µM in 1x siRNA buffer (Thermo
Scientific, Waltham, MA)) and incubating for 40 minutes at 37C with shaking on an Eppendorf
Thermomixer R. Resulting nanoparticles were analyzed for siRNA incorporation by resolution
on a 12% polyacrylamide gel followed by ethidium bromide staining. Dynamic light scattering
(DLS) and zeta potential measurements were performed on a Zeta Plus particle sizer
(Brookhaven Instruments, Newton, MA). Serum stability analysis was performed by incubating
freshly formed peptide/siRNA nanoparticles in 500 µg/mL human serum albumin (HSA, Sigma)
overnight followed by DLS and zeta potential measurements. Wet-mode atomic force
microscopy was performed by ARC Technologies (White Bear Lake, MN).
2.3.2 Analysis of p5RHH Disulfide Bond Formation
32
p5RHH was diluted to 5 mM in 20% DMSO and allowed to oxidize for 24 to 72 hours at
4°C. Disulfide bond formation was quantified using Ellman’s Reagent (ER) (20 mM stock in
buffer 8 (0.1 M NaH2PO4, 0.2 mM EDTA, pH 8)). Briefly, p5RHH was diluted 1 to 500 into ER
working solution (40 µM) and allowed to incubate at 37°C for 25 minutes. After the incubation,
UV absorbance was measured at 412 nm. A standard curve of freshly prepared L-cysteine was
used to allow quantification of thiol oxidation.
2.3.3 Cell Culture
B16-F10 (ATCC, Manassas, VA) cell lines were maintained under standard cell culture
conditions (37C and 5% CO2 in a humidified incubator) in DMEM (Gibco, Carlsbad, CA)
supplemented with 10% fetal bovine serum (Gibco). B16-F10 cells stably expressing GFP were
produced as follows: B16-F10 were transfected (Lipofectamine 2000, Invitrogen) with a fusion
of eGFP (pEGFP-N1, Clontech) and the PEST sequence from mouse ornithine decarboxylase
(S421-V461) in pEF6V5HisTOPO (Invitrogen); cells were selected for four rounds with cell
sorting by flow cytometry without antibiotic selection; an aliquot of cells was maintained in
continuous culture for a month without a noticeable change in eGFP expression.
2.3.4 siRNA Transfection
Cells were plated in 6-well plates 12 hours before transfection and cultured under
standard cell culture conditions. p5RHH/siRNA nanoparticles were prepared and incubated with
cells for 4 hours in a final volume of 1 mL Optimem I (Gibco) or appropriate media
supplemented with 10% FBS. Transfections were scaled accordingly for cells plated in 12-well
plates based on c

melittin-derived peptides for sirna delivery: mechanisms

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